How to measure rodent behavior and perform a neurological screen.

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1 An Organ Systems Approach to Experimental Targeting of the Metabolic Syndrome How to measure rodent behavior and perform a neurological screen. Fiona Harrison, PhD Department of Medicine Vanderbilt University Medical Center fiona.harrison@vanderbilt.edu

2 Introduction Mouse-centric lecture Apparatus for mice are typically the same as for rats but scaled down However, mice are not just little rats. Behavioral profiles differ

3 Why study behavior? Psychology - the basics of learning and memory. Fundamental rules apply to animals and humans Genetic manipulations - to assess phenotypes caused by genetic manipulations Experimental manipulations - to assess phenotypic changes caused by pharmacological, dietary or environmental manipulations

4 Colony Health Good practice Eliminate artefactual effects in your experiments (blindness, deafness, anosmic) e.g. Irwin screen or SHIRPA Appearance (grooming, fighting, coloration), weight, temperature, posture and gait, activity, neurological reflexes

5 Feeding behaviors Feeding = Survival Specialized circuits and mechanisms exist for hunger, thirst and satiation and all associated behaviors (e.g. activity and foraging) Insulin and glucose - gastrointestinal feedback - hindbrain - hypothalamus Many injected peptides can influence feeding Liao et al Nature Medicine

6 Feeding behaviors 24 hour consumption of standard chow and water (weight and volume) - Easy but not sensitive - Spillage, meal size, meal frequency, diurnal rhythm Restricted feeding (4 hours per day) - Easier to detect effects of short acting drugs Short access to highly palatable foods

7 Feeding behaviors Continuous monitoring can be assessed in automated chambers

8 Control diets Purina Standard lab chow Research Diets Inc. Basic control diet

9 Feeding behaviors Hedonic control Psychosocial factors Environment BRAIN Blood glucose Feeding behaviors/ Energy intake Digestive tract - Stomach; intestines; pancreas (ghrelin; GLP-1, PYY, CCK; insulin) Adipose tissue (leptin, adinopectin) Homeostatic feedback Hunger/satiety

10 Feeding behaviors Novel taste neophobia Social transmission of food preference > Preference for novel food

11 Locomotor activity

12 Locomotor activity Automated measurement by beam breaks Computer scored from top-view video Hand-scored by square crossings

13 Locomotor activity Habituation - reduced exploration with repeated exposure Within-trial and between-trials Activity response to drug e.g. scopolamine

14 Open field Anxiety Using locomotor activity chambers Preferential exploration of dark, enclosed, wall/corner areas Light-dark box

15 Elevated Plus/Zero Maze (EPM/EZM) Time spent in Open versus closed arms Should show preference for closed (dark)arms Entries into each arm Distance traveled Stretch-attend postures EZM has same exploration conflicts minus the central square

16 In the EPM mice can choose between exploration in open and closed areas

17 Depressive-like behaviors Porsolt or forced-swim - Behavioral despair indicated by time spent swimming - Be aware: Mice float, but rats sink Tail suspension test - Behavioral despair indicated by time spent immobile Behaviors improved by anxiolytic drugs

18 Abnormal behavior in db/db mouse

19 Abnormal behavior in db/db mouse a) Elevated Plus Maze b) Y-maze c) Porsolt forced swim c)

20 This image cannot currently be displayed. Motor skills Rotorod Motor ability and also motor/procedural learning across trials (cerebellum)

21 Gait Measured by specialized equipment or with (non-toxic) ink and paper Grip strength Measured by specialized equipment or by a rating scale Hill et al Nutritional Neuroscience

22 Inverted screen Motor skills

23 Motor skills Horizontal beam Wire hang Score according to performance - grip, posture correction

24 Learning and memory There are a lot of different tasks There are a lot of versions of each tasks There are a lot of bad behavioral experiments in the literature Explicit/declarative v Implicit/procedural

25 Y-maze - alternation A B C Working memory Mice prefer novelty To explore a new area, you must remember where you have already been

26 T-maze Hippocampal-dependent, spatial learning Rule: Non-match to sample Forced-choice trial followed by rewarded free-choice trial Advantage inclusion of time delays

27 Morris water maze First establish that your animal is physically capable of performing the task Rats are naturally better swimmers than mice Visible/Cued platform Above surface Flag/marker Then Hidden Platform Use extra-maze cues to locate the platform

28 Data interpretation A B C

29 Water maze is more stressful to mice - elicits greater levels of serum corticosterone High corticosterone correlates with poorer performance in MWM but not BM Harrison et al. (2009) Behavioural Brain Research 198,

30 Fear conditioning Learn and remember an association between an aversive experience and an environmental cue Memory of shock inferred from level of freezing on subsequent trials when exposed to the same arena Fear Conditioning 24 hours Test context Test conditioned stimulus 8 weeks treatment with leptin improved performance in APP/PSEN1 mice Greco et al. (2010) J Alz. Dis.

31 Fear conditioning

32 Dietary interventions Caloric restriction Improves cognition Increases longevity Increases arousal and activity Physiological changes High fat diet Impairs cognition Dietary change Change in consumption

33 Pharmacological interventions Must be able to cross blood brain barrier Routes of administration subcutaneous (s.c.) and intraperitoneal (i.p.) most common Dose required Therapeutic response window Side effects

34 Pharmacological interventions Leptin Ghrelin Neuropeptide Y Effects on activity Effects on consumption

35 Other considerations Noise Smells Experimenter Room size Room cleanliness Single housing Time of day Time of year Hey Ted, I would rather work with Tiffany

36 Labs I & II - Aims Gain confidence with handling of animals and restraint techniques Learn injection techniques See examples of behavioral tests See responses to pharmacological treatments

37 Lab I: Working with mice Location: Murine neurobehavioral lab 1:30-4:30 pm All groups: Mouse handling Group 1: Neuromuscular testing Group 2: Elevated plus maze Group 3: Locomotor activity If time allows - a tour of the rest of the behavior facility

38 Lab II: Working with rats Location: Murine neurobehavioral lab 1:30-5 pm Analisa Thompson-Gray & team Rat handling & injections (s.c., i.p.) Irwin screen with oxotremorin Rat rotorod

39 Recommended reading Crawley JN (2000) What's wrong with my mouse? : behavioral phenotyping of transgenic and knockout mice, Wiley-Liss, New York. Crawley JN (2008) Behavioral phenotyping strategies for mutant mice. Neuron 57,

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